pCDNA3.1(-)哺乳细胞表达载体质粒

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P0156 pCDNA3.1(-)哺乳细胞表达载体质粒

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别名: pCDNA3.1-
启动子: CMV promoter
复制子: pUC ori,F1 ori
终止子: BGH poly(A)signal
质粒分类: 哺乳系列质粒;哺乳表达质粒;pCDNA系列质粒
质粒大小: 5427bp
原核抗性: 氨苄青霉素(Ampicillin)
筛选标记: 新霉素(Neomycin)/遗传霉素(Geneticin/G418)
克隆菌株: DH5α等大肠杆菌
培养条件: 37℃,有氧,LB
表达宿主: 293T等哺乳细胞
培养条件: 37℃,5%CO2
诱导方式: 无需诱导
5'测序引物: pCDNA3.1-F(CTAGAGAACCCACTGCTTAC)
3'测序引物: pCDNA3.1-R(TAGAAGGCACAGTCGAGG)
备注: 需要添加起始密码子ATG
          pCDNA3.1(-)是来自pCDNA3的5.4kb载体,设计用于哺乳动物宿主中的高水平稳定和瞬时表达。大多数哺乳动物细胞可以进行高水平的稳定和非复制性瞬时表达。人类巨细胞病毒立即早期(CMV)启动子,用于在广泛哺乳动物细胞中的高水平表达。前向(+)和反向(-)方向上的多个克隆位点,以促进克隆用于选择稳定细胞系的新霉素抗性基因。在潜伏感染SV40或表达SV40大T抗原(例如COS-1,COS-7)的细胞系中的异常复制。
          pCDNA 3.1(-) is 5.4 kb vector derived from pcDN3 and designed for high-level stable and transient expression in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells. The vectors contain the following elements:  
          Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells  
          Multiple cloning sites in the forward (+) and reverse (C) orientations to facilitate cloning  
          Neomycin resistance gene for selection of stable cell lines  

          Episomal replication in cells lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS-1, COS-7)
          The control plasmid, pcDNA3.1/CAT, is included for use as a positive control for transfection and expression in the cell line of choice.
Use the following outline to clone and express your gene of interest in pcDNA 3.1. 
          1. Design  the multiple cloning  
          2.  Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on LB plates containing 100 g/ml ampicillin
          3.  Analyze your transformants for the presence of insert by restriction digestion. 
          4.  Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in the proper orientation. 
          5.  Transfect your construct into the mammalian cell line of interest using your own method of choice. Generate a stable cell line, if desired. 
          6.  Test for expression of your recombinant gene by western blot analysis or functional assay.
LOCUS       Exported                5427 bp ds-DNA     circular SYN 18-9-2016
DEFINITION  synthetic circular DNA
KEYWORDS    Untitled 10
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5427)
  TITLE     Direct Submission
  JOURNAL   Exported 2016-9-18 from MiaoLingPlasmid
            http://www.maiolingbio.com
FEATURES             Location/Qualifiers
     source          1..5427
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        235..614
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        863..881
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1027..1251
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1297..1725
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1739..2068
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      1919..2054
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2135..2929
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3103..3224
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3273..3289)
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    3297..3313
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(3321..3351)
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    3366..3387
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3675..4260)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(4431..5291)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5292..5396)
                     /gene="bla"
                     /note="AmpR promoter"
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga ctcgagcggc cgccactgtg ctggatatct gcagaattcc
      961 accacactgg actagtggat ccgagctcgg taccaagctt aagtttaaac cgctgatcag
     1021 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1081 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1141 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     1201 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     1261 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     1321 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1381 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1441 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1501 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1561 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1621 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1681 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     1741 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     1801 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     1861 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     1921 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     1981 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     2041 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     2101 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     2161 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     2221 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     2281 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     2341 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     2401 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     2461 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     2521 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     2581 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     2641 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     2701 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     2761 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     2821 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     2881 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     2941 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     3001 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     3061 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     3121 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3181 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     3241 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3301 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3361 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3421 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3481 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3541 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3601 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3661 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3721 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3781 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3841 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3901 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3961 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4021 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4081 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4141 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4201 gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4261 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4321 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4381 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4441 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4501 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4561 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4621 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4681 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4741 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4801 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4861 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4921 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4981 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5041 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5101 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5161 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5221 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5281 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5341 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5401 tttccccgaa aagtgccacc tgacgtc
//